Volume 2, Issue 6, Pages 602-612 (June 2008) High-Throughput Screening Assay for the Identification of Compounds Regulating Self- Renewal and Differentiation in Human Embryonic Stem Cells  Sabrina C. Desbordes, Dimitris G. Placantonakis, Anthony Ciro, Nicholas D. Socci, Gabsang Lee, Hakim Djaballah, Lorenz Studer  Cell Stem Cell  Volume 2, Issue 6, Pages 602-612 (June 2008) DOI: 10.1016/j.stem.2008.05.010 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 HTS Assay Development (A) HESCs (H9 line) maintained on Matrigel prior to 384-well plating exhibit typical undifferentiated cell morphology (phase contrast), express Oct4, and incorporate the S phase marker BrdU. (B) Cell titration on Matrigel-coated 384-well plates followed Alamar blue assay to determine the number of metabolically active cells (relative fluorescence units, RFU). Data are presented as mean ± SEM. Cell density refers to seeding density at the time of plating; Alamar blue is added at day 7, RFU is measured at day 8. (C) (Upper panel) Quantification of Oct4 signal after treatment of hESCs in 384-well plates with BMP4 (low control) and FGF2 (high control). (Lower panel) Corresponding immunocytochemical analysis for Oct4 and Nanog in BMP4 and FGF2-treated cells. AU corresponds to arbitrary units. Data are presented as mean ± SEM. (D) Schematic representation of HTS assay: undifferentiated hESCs were grown on Matrigel, dissociated into single cells using Accutase, and plated onto Matrigel-coated 384-well plates at a density of 6000 cells per well. After 48 hr, compounds (yellow) and high and low controls (red, FGF2; green, BMP4) were added for an additional 5 days. At day 7, automated immunocytochemistry for Oct4 was performed and signal intensity was quantified using automated laser-scanning confocal microscopy (GE InCell Analyzer 3000). The specific image shown corresponds to a culture well at day 7 after plating maintained in the presence of FGF2. (E) Control experiments in HTS conditions were carried out using IGF2 for similar effect as FGF2 and BMP2 and PD98059 for loss of Oct4 expression. Scale bar in (A) corresponds to 500 μm in (A) (left panel), 50 μm in (A) (right panel), and 100 μm in (C) (lower panels) and in (E). Cell Stem Cell 2008 2, 602-612DOI: (10.1016/j.stem.2008.05.010) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Results of 2880 Compound Library Screen (A) (Upper panel) Out of 89 compounds identified in the primary screen, 65 compounds were subjected to further dose-response analysis resulting in 22 confirmed hits. (Lower panel) Representative images illustrating effects of activator, inhibitor, and cytotoxic compounds. Scale bar corresponds to 80 μm. (B) Dose-response curves for a selected set of activators (left panel) and inhibitors (right panel). Data are expressed as Oct4 ratio of FGF2 versus BMP4-treated cells with FGF2 levels arbitrarily set at 100%. Levels of BMP4 and FGF2 treatment are marked in graph by dotted lines at 0% and 100%, respectively. (C) The 22 chemicals with dose-dependent effects on hESC fate could be grouped into several categories based on current clinical drug indications of the compounds. Significant enrichment in hit list compared to library composition was observed for the categories cardiac glycosides (p < 0.000001) and antiinflammatory compounds (p < 0.05). Cell Stem Cell 2008 2, 602-612DOI: (10.1016/j.stem.2008.05.010) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Small Molecules Promoting hESC Self-Renewal (A) Expression of Oct4 and Nanog upon treatment with the four activator compounds was confirmed in H9 and in H1 hESCs. (B) Repeated passaging of hESCs (H9) showed an increase in the percentage of undifferentiated colonies in presence of the self-renewal drugs, compared to HES medium alone (Ø). The graph represents the relative increase of undifferentiated colonies comparing HES medium alone (defined as 0%) versus MEF-CM supplemented with FGF2 (defined as 100%). Drug effects were tested at 50 and 100 μm as indicated. (C) Mouse ESC self-renewal was tested after three passages using an Oct4::eGFP BAC transgenic mouse ESC line. The percentage of eGFP+ cells was quantified by flow cytometry, and the levels of Oct4 cells achieved in mouse ESC medium alone (defined as Ø = 0%) were compared to those treated with LIF (defined as 100%). Drug effects were tested at 10 and 100 μM as indicated. (D) Quantification of the ratio of Oct4/DAPI staining under different culture conditions. Data are presented as mean ± SEM. Statistical analysis with ANOVA and post hoc Dunnett's test showed that the BMP4 group was significantly different from HES control (p < 0.001), while none of the other treatments were significantly different from control. (E) Representative images of Oct4 immunoreactivity in hESCs maintained with CM, FBP, THEA, and BMP4 over two passages. Note that BMP4 causes a rapid downregulation of Oct4. (F) Cell-cycle data for hESCs maintained with the self-renewal drugs or controls over two passages. Cell Stem Cell 2008 2, 602-612DOI: (10.1016/j.stem.2008.05.010) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Functional Response and Potential Downstream Factors in hESCs Exposed to Candidate Self-Renewal Compounds (A) TUNEL analysis of hESCs maintained in the presence of various compounds and under control conditions did not reveal significant differences among groups. Data are presented as mean ± SEM. (B) Pluripotency was assessed using directed differentiation protocols in hESCs maintained for three passages in the presence of compounds or control conditions. (C) Total of 1,145 transcripts out of 22,177 transcripts were differentially expressed during treatments of hESCs with CM, CM + bFGF, or self-renewal drugs compared to basal hESC medium alone. These genes were clustered, and a heat map for all conditions in duplicate or triplicates was established. (D) Venn diagram showing the number of transcripts differentially expressed in GTFX-treated cells or cells maintained in MEF-CM or in MEF-CM supplemented with FGF2 (CM + FGF2) versus cells maintained in basic hESC medium. Significance of overlap in GTFX versus CM was p < 10−10; GTXF versus CM + FGF2 was p < 10−16. (E) Venn diagram showing the number of transcripts differentially expressed in SNM-treated cells using the same parameters as in (D). Significance of overlap in SNM versus CM was p < 10−10 and p < 10−16 versus CM + FGF2. (F) Functional annotation analysis using DAVID platform (http://david.abcc.ncifcrf.gov; Dennis et al., 2003) illustrates overrepresentation of transcript categories within a given treatment group compared to control cultures. Significance and relative fold levels of enrichment are presented. Full description of the algorithms used, the definition of the categories, and list of all transcript members included per category are provided in Table S3 and in the Experimental Procedures. Cell Stem Cell 2008 2, 602-612DOI: (10.1016/j.stem.2008.05.010) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Small Molecules Driving hESC Differentiation (A) Expression of Oct4 was downregulated upon treatment with inhibitor compounds (left panels). Markers of differentiation were used to characterize lineage choice promoted by a given compound. Tretinoin (RA) showed strong induction of cytokeratin 18 (CK18), selegiline increased expression of Pax6 over time, and cymarin and sarmentogenin showed induction of Sox17 (right panels). These effects were established in the hESC line H9 and confirmed in H1. (B) (Upper panel) CK18 intensity signal was quantified using the confocal InCell Analyzer 3000 at day 7 of assay. (Lower panel) Under the same conditions, expression of Sox17 was quantified. Data are presented as mean ± SEM. Statistical significance was defined as: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ANOVA followed by Dunnett's test. (C) Percentage of Sox17+ cells under control conditions or in the presence of CYM, SRM, or ActivinA (positive control). (D) Representative images of Sox17 immunocytochemistry at the time of analysis used for quantification in (C). Scale bar in (D) corresponds to 10 μm. Cell Stem Cell 2008 2, 602-612DOI: (10.1016/j.stem.2008.05.010) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Potential Downstream Mediators in the Response of hESCs to Differentiation Compounds (A) A total of 6163 transcripts were differentially expressed during treatments of hESCs with KSR+DMSO or treatment with any of the inhibitor compounds compared with cells maintained in KSR medium alone. These genes were clustered, and a heat map for all conditions in duplicate or triplicates was established. (B) Venn diagram showing the number of transcripts differentially expressed in sarmentogenin (SRM), cymarin (CYM), or tretinoin (RA) versus cells maintained in KSR only. (C) The top ten upregulated and top five downregulated genes out of the total set of 22,177 transcripts are listed for the three drugs. Six out of the ten most regulated transcripts in CYM and SRM are identical. (D) Functional annotation analysis using DAVID platform (http://david.abcc.ncifcrf.gov/; Dennis et al., 2003) illustrates overrepresentation of transcript categories within a given treatment group compared to control cultures. Significance and relative fold levels of enrichment are presented. Full description of the algorithms used, the definition of the categories, and a list of all transcript members included per category are provided in Table S5 and in the Experimental Procedures. Cell Stem Cell 2008 2, 602-612DOI: (10.1016/j.stem.2008.05.010) Copyright © 2008 Elsevier Inc. Terms and Conditions